Expression of Inflammatory-Related NFκB Genes in Iranian Patients with Pterygium: A Case-Control Study

Pterygium is one of the most common eye conditions without any clear etiology. Some studies have suggested an association between sun exposure and pterygium, but others have proposed the role of genetic variations in its pathogenesis. To date, no study has investigated the association of inflammatory transcription factor, NFκB genes with pterygium in the Middle East. We examined the changes in expression of 3 inflammatory related NFκB1, NFκB2, and RELA genes in patients with pterygium. Thirty patients with pterygium and 30 age and sex-matched controls were enrolled in this case-control study. None of the participants showed any clinical signs of inflammation in their conjunctiva. Demographic information was obtained and the expression levels of three genes including NFκB1, NFκB2, and RELA were measured in their conjunctiva by real-time RT-PCR using gene-specific primers. Mean expression level of NFκB1, NFκB2 and RELA genes in patients were 2.4±0.3, 1.9± 0.5, and 1.8±0.4 times higher than normal subjects, respectively. Higher levels of gene expression were observed in individuals with more outdoor activity and sun exposure. Moreover, a significant correlation was observed between the expression levels of NFκB2 and RELA genes, suggesting a possible NFκB2- RELA heterodimer formation in patients with pterygium. This study has indicated a significant association between expressions of inflammatory-related NFκB1, NFκB2 and RELA genes, and pterygium. Further studies to verify the role of inflammation in the pathogenesis of pterygium, may provide new targets for managing pterygia.

terygium is a common ocular disorder characterized by the growth of a wing-like connective tissue on the cornea (1) that can cause irritation, foreign body sensation, and conjunctival hyperemia. It also induces astigmatism and affects visual quality (1,2). Pterygium occurrence is generally high in semi-dry regions, and its prevalence was reported to be 2% according to a cohort study in Iran the prevalence of pterygium in the urban population was 9.4% and 2.9% in one and both eyes, respectively (4).
Exposure to sunlight and genetic factors are the two main proposed contributors in the initiation and development of pterygium (5)(6)(7). Sunlight can induce inflammation, DNA damage, and secretion of growth factors, resulting in pterygium (8). So far, several reports have proposed the role of inflammation in pathogenesis, and post-surgery outcomes in pterygia (9)(10)(11). The nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) family members including NF-κB1 (p50), NF-κB2 (p52), RELA (p65), RELB and C-REL, are the main mediators in initiating inflammation by inducing pro-inflammatory agents expression (12).
Formation and activation of different dimers of NF-κB members mediate specific target gene expression in response to various stimuli (13).
Various biological processes such as proliferation, apoptosis, differentiation, and migration are regulated through the NF-κB pathway with expression of more than 150 genes (14). Of the important relevant genes in pterygium are epithelial neutrophil activating peptide 78 (CXCL5), monocyte chemotactic protein 1 (CCL2), and matrix metalloproteinases (MMPs) (15). A few studies suggested the role of NF-κB in cellular responses to UV damage and hyperosmotic stress (16,17) that might have a role in pterygium.
Siak et al., for the first time reported NF-κB pathway activation in pterygium in Singaporean patients (18). Another study at 2017 showed elevated levels of local pro-inflammatory cytokine and nitric oxide responses in pterygium of Algerian patients (19). Recently, a few studies have concluded that despite the role of NF-κB family members as an initiator of inflammation, their inhibition after the initial inflammatory insult, may prolong the process of inflammation and delay tissue repair (20,21).
In this study, we evaluated the probable associations between the mean expression levels of NFκB1, NFκB2 and RELA genes in the pterygium.
Amongst all family members, these three proteins are more prevalent in activated form of NFκB heterodimers (12,22). Dysregulation or aberrant expression of NFκB proteins were shown to be linked with neoplasms, autoimmune and inflammatory disorders (23)(24)(25)(26). Therefore, the aim of this study was to examine the expression of these inflammatory-related genes in patients with pterygia in a case-control study.    Mean sun exposure between the two groups was evaluated by student t-test and was statistically significant (P= 0.002).

Results
The relative genes' expressions in patients versus controls are shown in figure 1.
Quantification of NFκB1, NFκB2 and RELA expression showed a significant difference between cases and controls (P˂ 0.0001) ( Table 3).
Moreover, a significant correlation was found between NFκB2 and RELA expressions in patients as verified by Pearson's correlation test (2-tailed).
No statistically significant correlation was found between expressions of NFκB2 and NFκB1 or RELA and NFκB1.

Discussion
A few studies have suggested that pterygium should be classified as a degenerative process (28), while others consider it as an unusual growth disorder (29,30), or a dysfunction in limbal stem cells (31).
The results of epidemiologic experiments revealed some associations between pterygium with other sun-related eye disorders, such as cataract, basal cell carcinoma, and spheroidal degeneration (31)(32)(33)(34). In vitro experiments showed the major role of ultra violet ray in initiating pterygium (35)(36)(37). However, more studies are needed to verify the role of inflammation in the etiology of pterygium.
We also found a positive correlation between NFκB2 and RELA genes in the pterygium patients.
These two proteins can form NFκB heterodimer. In conclusion, in this study, by measuring